The presence of food spoilage organisms and pathogens in foods is a major concern to the food processing industry, government regulatory agencies and food consumers. Foodborne pathogens have been responsible for several food poisoning outbreaks, some of which have resulted in serious illness and death. In addition, the presence of pathogenic organisms in foods has led to numerous product recalls, product losses, and considerable negative publicity to the food industry. For example, a report of a case of listeriosis associated with the consumption of turkey franks provided direct evidence of the infection by Listeria monocytogenes linked to poultry products (Barnes et al., Morbid. Mortal. Weekly Rep. 38:267-268 (1989)). It has also been shown that L. monocytogenes occurs commonly in seafoods, poultry, and meats including cured and fermented meats. See, for example, Buchanan et al, Appl. Environ. Microbiol. 55:599-603 (1989); Bailey et al., J. Food Prot. 52:148-150 (1989); M. Gitter, Vet. Res. 99:336 (1976); and Farber et al., Can. Inst. Food Sci. Technol. J. 21:430-434 (1988).
Lactic acid bacteria are known to produce inhibiting substances such as hydrogen peroxide, antibiotics and bacteriocins. These bacteria are also capable of lowering the oxidation/reduction potential of the growth medium, and may compete with other lactic acid bacteria or pathogens and spoilage organisms for essential nutrients.
Lactic acid bacteria, including Lactobacillus species, and Pediococcus species, used as fermentation starter cultures, have been screened and shown to produce bacteriocins. The spectrum of bacteriocin activity against various microorganisms, including Listeria monocytogenes and other bacterial strains, has also been assessed (as cited in Richter et al., Abstract P8, 89th Annual Meeting, American Society of Microbiology, New Orleans, LA, May 14-18 (1989); and Richter, K. S., M. S. Thesis entitled "Physical and Genetic Properties of a Bacteriocin Produced by a Pediococcus sp. Active Against Listeria monocytogenes, University of Nebraska-Lincoln (1989), the disclosure of which are incorporated by reference herein). For example, pediocin PA-1 produced by Pediococcus acidilactici PAC 1.0 has been found to inhibit a wide spectrum of gram positive organisms, including several genera of lactic acid bacteria and various food isolates of Listeria monocytogenes. P. pentosaceus FBB63 also inhibited L. monocytogenes, although to a lesser extent. Other bacteriocins of the genus Pediococcus and their spectrum of activity have also been characterized, including pediocin AcH by P. acidilactici H, and pediocin A by P. pentosaceus FBB61, L7230 and FBB63 (as cited in K. S. Richter, M. S. Thesis, page 41-43, Univ. Neb.-Lincoln (1989 )). The antimicrobial activity of pediocin AcH, was tested against several pathogenic and food spoilage bacteria, including Listeria monocytogenes (Bhunia et al., J. Appl. Bacteriol. 65:261-268 (1988)).
The production of bacteriocin by various strains of Pediococcus species has been associated with the presence of plasmid DNA D. Graham and L. McKay, Appl. Envir. Microb. 50:532-534 (1985); C. Gonzalez and B. Kunka, Appl. Envir. Microb. 53:2534-2538 (1987)). Bacteriocins associated with plasmid DNA from strains of Pediococcus acidilactici and P. pentosaceus have been shown to inhibit Listeria monocytogenes, Streptococcus faecalis and Leuconostoc mesenteroides (Hoover, et al., J. Food Protection 51:29-31 (1988)).
Several ways of inhibiting Listeria and other foodborne pathogens in food products using bacteriocins have been reported. For example, bacteriocins isolated from lactic acid bacteria have been separately added to food products to inhibit foodborne organisms. U.S. Pat. No. 4,929,445 to Vandenbergh et al. (issued May 19, 1990; filed Jan. 25, 1988) discloses a method of inhibiting Listeria monocytogenes in unspoiled foods, such as milk-based cheeses, ice cream, meats and fish, by adding bacteriocin derived from Pediococcus acidilactici as a dried powder to inhibit the Listeria at a pH range of about pH 4-9. Pucci et al disclose that where a dried powder of bacteriocin PA-1 produced by Pediococcus acidilactici was used to inhibit Listeria monocytogenes, it was found that the Listeria was inhibited due to a synergistic effect of lactic acid and bacteriocin (Appl. Envir. Microb. 54:2349-2353 (1988)). Since the production of bacteriocin preparations can be time-consuming and costly, it is desired that a system be developed in which viable cells of bacteriocin-producing organisms may be directly added to a food substance to provide inhibition of pathogens and food spoilage organisms.
In food fermentations, bacteriocin-producing lactic acid bacteria have been used as fermentation starter cultures for fermenting meat and providing bacteriocins to inhibit growth of pathogens and spoilage organisms. For example, bacteriocin-producing Pediococcus species were used as a fermentative starter culture to inhibit Listeria monocytogenes in fermented summer sausage (Richter, K. S., M. S. Thesis entitled "Physical and Genetic Properties of a Bacteriocin Produced by a Pediococcus sp. Active Against Listeria monocytogenes, University of Nebraska-Lincoln (1989)). The preservation of the cured, dried, fermented sausage from spoilage and pathogenic microorganisms was due to a number of factors, including low water activity, sodium chloride, sodium nitrite, and low pH due to the production of lactic acid by the starter culture organisms. However, while growth may be suppressed during fermentation and the drying process, these organisms may survive in the finished product. Therefore, there is a need for a means of inhibiting growth of foodborne pathogens and food spoilage organisms in fully processed, and/or fermented or cured foods.
Psychrotrophic bacteria can grow at temperatures as low as 4.degree. C., so that even refrigeration offers relatively minimal protection if a food product is contaminated with these organisms. Viable cells of lactic acid bacteria which produce hydrogen peroxide without fermentation have been used to inhibit food-borne human pathogen or spoilage microorganisms in food during refrigerated storage. Lactobacillus bulgaricus and L. lactis inhibited psychrotrophic bacteria, Pseudomonas fragi and psychrotroph MC-60N, a Gram negative rod isolated from raw milk, in ground beef stored at 5.degree. C. by production of hydrogen peroxide. (S. E. Gilliland and M. L. Speck, J. Food Science 40:903-905 (1975)). In ground and mechanically deboned poultry meat refrigerated at 3.degree. C., resting cells of meat starter cultures of Pediococcus cerevisiae and/or Lactobacillus plantarum were shown to inhibit psychrotrophic bacteria (Raccach et al., J. Food Science 44:43-46 (1979)). In refrigerated, packaged processed food, viable cells of a Lactobacillus which produced hydrogen peroxide without fermentation were used to inhibit food-borne pathogens and spoilage microorganisms (U.S. Pat. No. 4,874,704 to Boudreaux et al. (issued Oct. 17, 1989; filed Mar. 14, 1988)). A composition of a lactic acid-producing bacteria, such as Streptococcus lactis subsp. diacetylactis, which produces a diacetyl flavor, and a Pediococcus which generates hydrogen peroxide, such as Pediococcus pentosaceus, were disclosed for inhibiting psychrotrophic bacteria in refrigerated milk or cream-based products, without fermentation or increase in cell count (U.S. Pat. No. 4,880,743 to Matrozza et al. (issued Nov. 14, 1989; filed Jul. 27, 1988)). However, since hydrogen peroxide can adversely affect the organoleptic characteristics of a food substance, there is a need for a non-destructive means of controlling psychrotrophs in meat and other food products under refrigeration conditions, by organisms which do not produce hydrogen peroxide in significant amounts in the food product.
Lactobacillus sake LL6706, a Lactobacillus species which produces the bacteriocin sakacin A, was found to inhibit Listeria monocytogenes in refrigerated meat. A bacteriocin effect was observed in comminuted cured pork at 15.degree. C. and pH 5.7, but not in pork of pH 6.3-6.4, nor in pasteurized minced meat held 6 days at 8.degree. C. (Lucke et al., FEMS Microb.Rev. 87:P85 (Abstract E3) (1990)). It was found that the use of L. sake as a "protective" culture in meats was limited by several factors including the slow diffusion rate of sakacin A to target cells, the inactivation of this bacteriocin over time, and the lack of sensitivity of all strains of L. monocytogenes to sakacin A.
Non-carbohydrate fermenting lactic acid bacteria have also been used to inhibit spoilage of lactose-containing food products. For example, cells of a non-lactose fermenting Streptococcus lactis have been added to lactose-containing foods, particularly milk products, to inhibit spoilage (U.S. Pat. No. 4,599,313 to Gonzalez, C. F. (issued Jul. 8, 1986)).
Therefore, an object of the invention is to provide a system in which an organism capable of producing bacteriocin will inhibit the growth of pathogens and spoilage organisms in foods by producing bacteriocin without cell growth and/or fermentation. Another object is to provide a non-destructive method of inhibiting pathogens and spoilage organisms in foods using living cells of lactic acid bacteria wherein the organoleptic properties of the food are not changed as a result of cell growth and/or fermentation by the bacteriocin-producing organism. A further object is to provide a method in which living cells of lactic acid bacteria are combined with a food substance to inhibit the growth of pathogens and spoilage organisms by providing inhibiting amounts of bacteriocin. Yet another object is to provide a food mixture containing living cells of lactic acid bacteria and a food substance, wherein the lactic acid bacteria inhibit the growth of pathogens and/or spoilage organisms by producing bacteriocin without cell growth and/or fermentation.